In recent decades, evidence has been accumulating showing the important role of urokinase-type plasminogen activator (uPA) in growth, invasion, and metastasis of malignant tumours. The evidence comes from results with animal tumour models and from the observation that a high level of uPA in human tumours is associated with a poor patient prognosis. It therefore initially came as a surprise that a high tumour level of the uPA inhibitor plasminogen activator inhibitor-I (PAI-I) is also associated with a poor prognosis, the PAI-I level in fact being one of the most informative biochemical prognostic markers. We review here recent investigations into the possible tumour biological role of PAI-I, performed by animal tumour models, histological examination of human tumours, and new knowledge about the molecular interactions of PAI-I possibly underlying its tumour biological functions. The exact tumour biological functions of PAI-I remain uncertain but PAI-I seems to be multifunctional as PAI-I is expressed by multiple cell types and has multiple molecular interactions. The potential utilisation of PAI-I as a target for anti-cancer therapy depends on further mapping of these functions.

PAI-1 is a Mr ~50,000 glycoprotein, which is the primary physiological inhibitor of the two plasminogen activators uPA and tPA. PAI-1 belongs to the serpin protein family. Studies of PAI-1 have contributed significantly to the elucidation of the protease inhibitory mechanism of serpins, which is based on a metastable native state becoming stabilised by insertion of the RCL into the central beta-sheet A and formation of covalent complexes with target proteases. In PAI-1, this insertion can occur in the absence of the protease, resulting in generation of a so-called latent, inactive form of the protein. PAI-1, in its active state, also binds to the extracellular protein vitronectin. When in complex with its target proteases, it binds with high affinity to endocytosis receptors of the low density receptor family.

IntroductionThrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy characterized by a schistocytic hemolytic anemia and consumptive thrombocytopenia with varying degrees of neurologic and renal impairment. The presence of ultralarge von Willebrand factor (VWF) multimers in the plasmas of patients with chronic relapsing TTP during remission that disappear during an attack led to the implication of these multimers in the pathogenesis of the platelet-rich, fibrin-poor thrombi that occlude arterioles and are the hallmark of this disorder. 1 The VWF-cleaving metalloproteinase, ADAMTS13, hydrolyzes the ultralarge VWF multimers as they emerge from endothelial cells and undergo conformational strain due to shear stress in arterioles and capillaries. 2 A severe deficiency of VWF-cleaving proteinase activity is associated with the pathogenesis of congenital TTP (the SchulmanUpshaw syndrome). 3,4 A number of mutations, spread throughout the ADAMTS13 gene, have been reported in association with congenital TTP. 3,[5][6][7][8][9] The CUB domains at the C-terminus of ADAMTS13 are of uncertain physiologic relevance. The findings that the CUB domains are not required for VWF-cleaving proteinase activity measured under static conditions 10,11 and that certain strains of mice have a variant form of murine ADAMTS13 that lacks the CUB domains (together with the seventh and eighth thrombospondin type 1 (TSP-1) repeats) support the premise that these domains are dispensable in vivo. 12 However, a report that peptides from the CUB domains inhibit VWF-cleaving proteinase activity under flow, but not static, conditions suggests a functional role for these domains. 13 We studied a patient with congenital TTP who is a compound heterozygote for the Thr196Ile mutation in the metalloproteinase domain and the insertion of a nucleotide (A) at 4143-4144. This insertion results in a frameshift in the second CUB domain and loss of the last 49 amino acids of the protein. The CUB mutation had little effect on the specific activity of the enzyme but its secretion from COS-7 cells was impaired. Study design Case historyWe investigated an 8-year-old white girl with congenital TTP. Diagnosis was made when she was 18 months of age; she receives a plasma infusion every 2 to 3 months when her platelet count falls below 30 ϫ 10 9 /L. Assay of plasma VWF-cleaving proteinase activityAn assay based on the collagen-binding activity of digested VWF 14 was used to measure the VWF-cleaving proteinase activity in the plasma of the patient (during remission) and her family. ADAMTS13 sequence analysisDNA was extracted from peripheral blood leukocytes and all the exons and intron-exon boundaries of the ADAMTS13 gene were amplified by polymerase chain reaction (PCR) and sequenced. 5 The study was conducted with parental consent and institutional ethics approval. Transient expression of the ADAMTS13 mutantThe 4143insA mutant of the ADAMTS13 cDNA 15 was created using PCR-based mutagenesis. The wild-type and mutant constructs were subcloned into the mam...

To find new principles for inhibiting serine proteases, we screened phage-displayed random peptide repertoires with urokinase-type plasminogen activator (uPA) as the target. The most frequent of the isolated phage clones contained the disulfide bridgeconstrained sequence CSWRGLENHRMC, which we designated upain-1. When expressed recombinantly with a protein fusion partner, upain-1 inhibited the enzymatic activity of uPA competitively with a temperature and pH-dependent K i , which at 25°C and pH 7.4 was ϳ500 nM. At the same conditions, the equilibrium dissociation constant K D , monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was ϳ400 nM. By an inhibitory screen against other serine proteases, including trypsin, upain-1 was found to be highly selective for uPA. The cyclical structure of upain-1 was indispensable for uPA binding. Alanine-scanning mutagenesis identified Arg 4 of upain-1 as the P 1 residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P 1 , P 2 , P 3 , P 4 , and the P 5 residues of upain-1. Substitution with alanine of the P 2 residue, Trp 3 , converted upain-1 into a distinct, although poor, uPA substrate. Upain-1 represents a new type of uPA inhibitor that achieves selectivity by targeting uPA-specific surface loops. Most likely, the inhibitory activity depends on its cyclical structure and the unusual P 2 residue preventing the scissile bond from assuming a tetrahedral geometry and thus from undergoing hydrolysis. Peptide-derived inhibitors such as upain-1 may provide novel mechanistic information about enzyme-inhibitor interactions and alternative methodologies for designing effective protease inhibitors.Serine proteases of the trypsin family (clan SA) have many physiological and pathophysiological functions. There is therefore extensive interest in generating specific inhibitors to be used for pharmacological interference with their enzymatic activity. Moreover, serine proteases are classical subjects for studies of catalytic and inhibitory mechanisms.Serine protease-catalyzed peptide bond hydrolysis proceeds through a tetrahedral transition state formed by a nucleophilic attack on the carbonyl group of the substrate P 1 amino acid by the hydroxyl group of Ser 195 (using the chymotrypsin template numbering (1)), with His 57 and Asp 102 acting as a charge relay system. The protonated His 57 functions as a general acid to facilitate collapse of the tetrahedral intermediate that is stabilized through interactions at the oxyanion hole and main chain ␤-strand-type hydrogen bonds between the P 1 -P 3 and P 2 Ј amino acids of the substrate and residues within the polypeptide binding cleft, as well as specific contacts within the S 1 , S 2 , S 3 , S 1 Ј, and S 2 Ј pockets, which bind respective side chains of the P 1 , P 2 , P 3 , P 1 Ј, and P 2 Ј residues (for reviews, see Refs. 2 and 3). Substrate specificity is governed by the fit of the P residues into their corresponding S-pockets as ...